It is estimated that over 170 million people worldwide are infected with the Hepatitis C virus (HCV). With an estimated human sero-prevalence of 3% globally, HCV is the major cause for most cases of non-A, non-B hepatitis, (Alberti, A. et al., J. Hepatology 31., (Suppl. 1): 17-24, 1999). While the symptoms of acute hepatitis subside in some patients, at least 85% of HCV infections become chronic, and 20% of those infected develop liver cirrhosis. There is less than a 50% survival rate at four years post cirrhosis diagnosis. Chronic HCV infection is also associated with increased incidence of hepatocellular carcinoma.
HCV is a positive-stranded RNA virus whose genome encodes a polyprotein of approximately 3000 amino acids. This precursor protein is processed into at least 10 viral structural and nonstructural proteins: C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (Blight, K. J., et al., Antiviral Ther. 3, Suppl. 3: 71-81, 1998). HCV nonstructural (NS) proteins are derived by proteolytic processing of the polyprotein and are presumed to provide the essential catalytic machinery for viral replication.
NS3 is an approximately 68 Kda protein, and has both an N-terminal serine protease domain and an NTPase/helicase domain at its C-terminus. It has been shown that the NS4A protein serves as a co-factor for the serine protease activity of NS3. NS3 functions as a proteolytic enzyme that is involved in processing the viral polyprotein to liberate other nonstructural proteins necessary for HCV replication, and is a viable therapeutic target for antiviral chemotherapy. Internal self-cleavage of NS3 in its helicase domain also occurs, and this activity is dependent on both NS4a and NS3 protease activity.
Current methods for assaying inhibition of HCV protease rely on the indirect measurement HCV NS3 protease activity by measuring viral RNA replication in a Huh-7 replicon assay, often by utilizing secondary “reporters.” For example, luciferase is often utilized as a reporter to indicate the number of viral RNA molecules in a cell. Inhibition of NS3 protease activity leads to inhibition of viral replication, over time, and can be measured by decreased luciferase expression. However, to date, the direct measurement of HCV protease into its self-cleavage products has not been reported.
No vaccines are available for HCV, and the established therapy of interferon treatment is effective in only 15-20% of patients (Weiland, O., FEMS Microbiol. Rev. 14: 279-88, 1994), and has significant side effects (Walker, M. A., et al., DDT 4: 518-29, 1999; Moradpour, D., et al., Eur. J. Gastroenterol. Hepatol. 11: 1199-1202, 1999). While the current standard of care, pegylated interferon α in combination with ribavirin, is more efficacious and appears to decrease hepatocellular carcinoma in patients with HCV-related cirrhosis (Hung, C. H., et al., J Viral Hepatitis 13(6): 409-414, 2006), this treatment has also been shown to produce side effects such as thyroid dysfunction (Huang, J. F., et al., J Viral Hepatitis 13(6): 396-401, 2006).
The poor prognosis for patients suffering from HCV infection and the current lack of effective, approved treatments, highlights the overwhelming need for new inhibitors of HCV NS3 protease and methods for identifying the same.